Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.11.2 (prolyl hydroxylase)
 1,814 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.
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PMID:Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-alpha-prolyl-4-hydroxylases. 1510 34

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation.
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PMID:Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors. 1515 61

The HIFs (hypoxia-inducible factors) are a family of heterodimeric transcription factors essential for the adaptation of cells to reduced oxygen supply. Three human PHDs (prolyl hydroxylase domain proteins, PHD1-PHD3) initiate oxygen-dependent degradation of HIF-alpha-subunits in normoxia. RNA interference directed against PHD2, but not PHD1 or PHD3, is sufficient to stabilize HIF-1alpha in normoxia. Therefore PHD2 is regarded as the main cellular oxygen sensor. PHD2 itself is up-regulated by hypoxia and may thus limit hypoxic signalling. By sequence analysis, we predicted a promoter approx. 3.5 kb 5' of the translation start codon and a second promoter located in a CpG island immediately upstream of the coding sequence. A consensus HIF-1-binding site that is conserved in the murine phd2 gene was detected in the CpG island. By electrophoretic mobility-shift assay, we demonstrated binding of HIF-1 to the putative HIF-1-binding site. In luciferase reporter vectors, the isolated upstream promoter was inactive in all cell lines tested unless 200 bp were deleted at the 3'-end. The downstream promoter was active and induced by hypoxia. In reporter vectors containing both promoter sequences, luciferase activity was equal to vectors containing only the downstream promoter. In cells transfected with a vector containing both promoters, a single luciferase transcript was detectable. This transcript had the same length as transcripts from a vector containing the downstream promoter only. We conclude that the phd2 gene is transcribed exclusively from the downstream promoter that contains a functional hypoxia-responsive, cis-regulatory element. Our results establish that PHD2 is a direct HIF target gene.
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PMID:Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element. 1556 75

Oxygen-dependent proteolysis is the primary means of regulating the hypoxia-inducible factor (HIF) family of transcription factors. The alpha-subunit of HIF factor 1 (HIF-1) contains two highly conserved oxygen-dependent degradation domains (402 ODD and 564 ODD), each of which includes a proline that is hydroxylated in the presence of oxygen, allowing the von Hippel-Lindau (VHL) E3 ubiquitin ligase to interact and target HIF-1alpha to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1alpha under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1alpha are unknown. Here we show that the two ODDs of HIF-1alpha have independent yet interactive roles in the regulation of HIF-1alpha protein turnover, with the relative involvement of each ODD depending on the levels of oxygen. Using hydroxylation-specific antibodies, we found that under conditions of normoxia proline 564 is hydroxylated prior to proline 402, and mutation of proline 564 results in a significant reduction in the hydroxylation of proline 402. Mutation of proline 402, however, has little effect on the hydroxylation of proline 564. To determine whether the more rapid hydroxylation of the proline 564 under conditions of normoxia is due to a preference for the particular sequence surrounding proline 564 or for that site within the protein, we exchanged the degradation domains within the full-length HIF-1alpha protein. In these domain-swapping experiments, prolyl hydroxylase domain 1 (PHD1) and PHD2 preferentially hydroxylated the proline located in the site of the original 564 ODD, while PHD3 preferred the proline 564 sequence, regardless of its location. At limiting oxygen tensions, we found that proline 402 exhibits an oxygen-dependent decrease in hydroxylation at higher oxygen tensions relative to proline 564 hydroxylation. These results indicate that hydroxylation of proline 402 is highly responsive to physiologic changes in oxygen and, therefore, plays a more important role in HIF-1alpha regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1alpha has a distinct activity in controlling HIF-1alpha stability in response to different levels of oxygenation.
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PMID:Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1 alpha. 1602 80

Cellular response to limiting oxygen levels is managed, in part, by the transcription factor hypoxia-inducible factor 1 (HIF-1), and the prolyl hydroxylase (PHD) family of oxygen-requiring enzymes. In the process of analyzing the expression of PHD3, we observed the presence of two alternatively processed PHD3 transcripts, designated PHD3Delta1 and PHD3Delta4 . The expression of both PHD3 and PHD3Delta1 was observed in all tissues and cell lines tested, although the expression of the novel PHD3Delta4 appeared to be restricted to primary cancer tissues. The function of PHD3Delta4 was assessed in transfection experiments showing a preserved prolyl hydroxylase activity. We would submit that PHD3 variants generated by alternative splicing may be intrinsically involved in the complex system of oxygen sensing.
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PMID:An alternatively spliced transcript of the PHD3 gene retains prolyl hydroxylase activity. 1647 74

The heterodimeric transcription factor HIF (hypoxia-inducible factor) is central to the regulation of gene expression by oxygen. Three oxygen-dependent prolyl hydroxylase enzymes [PHD1 (prolyl hydroxylase domain 1), PHD2 and PHD3] control the abundance of HIF. In the presence of oxygen, they hydroxylate specific proline residues in HIF-alpha, allowing recognition by pVHL (von Hippel-Lindau protein) and subsequent ubiquitylation and proteasomal destruction. The precise roles and regulation of these enzymes are therefore of particular importance in understanding the physiological and pathological responses to hypoxia. In the present study, we define the existence of two species of PHD1 and provide evidence that they are generated by alternative translational initiation. We demonstrate that these alternative forms are both biologically active with similar HIF prolyl hydroxylase activity but that they differ in their responses to oestrogen, cell confluence and proteasomal inhibition. We show that the two PHD1 species are subject to proteolytic regulation but differ markedly in their protein stability. Though each isoform has the potential to interact with members of the Siah (seven in absentia homologue) ubiquitin ligase family, genetic studies indicated that other proteolytic mechanisms are responsible for control of stability under the conditions examined. The data define the existence of a further level of control in the pathway that regulates cellular responses to hypoxia.
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PMID:Characterization of different isoforms of the HIF prolyl hydroxylase PHD1 generated by alternative initiation. 1650 23

Hypoxia-inducible factor (HIF)-alpha subunits (HIF-1alpha, HIF-2alpha and HIF-3alpha), which play a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH), are regulated through post-translational hydroxylation by their three prolyl hydroxylase domain-containing proteins (PHD1, PHD2 and PHD3). PHDs could also be regulated by HIF. But differential and reciprocal regulation between HIF-alpha and PHDs during the development of HPH remains unclear. To investigate this problem, a rat HPH model was established. Mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. HIF-1alpha and HIF-2alpha mRNA increased slightly after 7 d of hypoxia, but HIF-3alpha increased significantly after 3 d of hypoxia. The protein expression levels of all three HIF-alpha were markedly upregulated after exposure to hypoxia. PHD2 mRNA and protein expression levels were upregulated after 3 d of hypoxia; PHD1 protein declined after 14 d of hypoxia without significant mRNA changes. PHD3 mRNA and protein were markedly upregulated after 3 d of hypoxia, then the mRNA remained at a high level, but the protein declined after 14 d of hypoxia. In hypoxic animals, HIF-1alpha proteins negatively correlated with PHD2 proteins, whereas HIF-2alpha and HIF-3alpha proteins showed negative correlations with PHD3 and PHD1 proteins, respectively. All three HIF-alpha proteins were positively correlated with PHD2 and PHD3 mRNA. In the present study, HIF-alpha subunits and PHDs showed differential and reciprocal regulation, and this might play a key pathogenesis role in hypoxia-induced pulmonary hypertension.
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PMID:Differential and reciprocal regulation between hypoxia-inducible factor-alpha subunits and their prolyl hydroxylases in pulmonary arteries of rat with hypoxia-induced hypertension. 1676 Nov 1

PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxia-inducible factor alpha subunits (HIF-alpha). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2(-/-) embryos died between embryonic days 12.5 and 14.5 whereas Phd1(-/-) or Phd3(-/-) mice were apparently normal. In Phd2(-/-) mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-alpha protein levels in the placenta and the embryo proper, there was no specific HIF-alpha increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.
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PMID:Placental but not heart defects are associated with elevated hypoxia-inducible factor alpha levels in mice lacking prolyl hydroxylase domain protein 2. 1696 70

The mechanisms controlling the expression of the gene encoding for the hormone erythropoietin (EPO) are exemplary for oxygen-regulated gene expression. In humans and other mammals, hypoxia modulates EPO levels by increasing expression of the EPO gene. An association between polycythaemia and people living at high altitudes was first reported more than 100 years ago. Since the identification of EPO as the humoral regulator of red blood cell production and the cloning of the EPO gene, considerable progress has been made in understanding the regulation of EPO gene expression. This has finally led to the identification of a widespread cellular oxygen-sensing mechanism. Central to this mechanism is the transcription factor complex hypoxia-inducible factor (HIF)-1. The abundance and activity of HIF-1, a heterodimer of an alpha- and beta-subunit, is predominantly regulated by oxygen-dependent post-translational hydroxylation of the alpha-subunit. Non-heme ferrous iron containing hydroxylases use dioxygen and 2-oxoglutarate to specifically target proline and an asparagine residue in HIF-1alpha. As such, the three prolyl hydroxylases (prolyl hydroxylase domain-containing protein (PHD) 1, PHD2 and PHD3) and the asparagyl hydroxylase (factor inhibiting HIF (FIH)-1) act as cellular oxygen sensors. In addition to erythropoiesis, HIF-1 regulates a broad range of physiologically relevant genes involved in angiogenesis, apoptosis, vasomotor control and energy metabolism. Therefore, the HIF system is implicated in the pathophysiology of many human diseases. In addition to the tight regulation by oxygen tension, temporal and tissue-specific signals limit expression of the EPO gene primarily to the fetal liver and the adult kidney.
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PMID:Hypoxia-induced erythropoietin production: a paradigm for oxygen-regulated gene expression. 1700 76

Hypoxia is an important physiological condition during embryonic development. Hypoxia-inducible factor (HIF) is the mediator of hypoxic response of cells. The prolyl hydroxylase (PHD) of HIF plays a key role in stabilizing of HIF and the oxygen homeostasis of organisms. In this study, we isolated two PHD proteins, PHD45 and PHD28, and characterized them during the embryonic development of Xenopus laevis, which is an excellent model for embryonic development because of the ease of embryonic manipulation and the feasibility of transgenesis. Based on amino acid sequences, Xenopus PHD45 and PHD28 were homologous with human PHD2 and PHD3, respectively. In embryonic development, PHD45 expression was complementary to that of PHD28. xHIF-1alpha protein level was at a maximum around stage 20 when expression of PHD45 disappeared, while expression of PHD28 reached a maximum at stage 20, suggesting that PHD28 is inducible by HIF-1alpha. Recently, Siah2 was found to be an ubiquitin ligase of PHD proteins and to regulate degradation of PHD proteins. Over-expression of xSiah2 decreased PHD45 but not PHD28 and caused the small-eye phenotype of Xenopus. Additional over-expression of PHD47 rescued the abnormality caused by xSiah2, suggesting that the level of expression or activity of PHD proteins is important to the maintenance of homeostasis in embryonic development.
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PMID:Isolation of Xenopus HIF-prolyl 4-hydroxylase and rescue of a small-eye phenotype caused by Siah2 over-expression. 1730 83


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